ICAT (Isotope-coded affinity-tag-based protein profiling)

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ICAT (1) is a gel-free MS based technique that is used to compare up to 2 samples. This approach utilizes a novel chemical reagent which consists of a thio-reactive group (labeling cysteines), an isotopic linker region (9 x C13 residues for the heavy tag), and an acid cleavable biotin moiety (for affinity based purification). Samples are labeled with “heavy” (C13) (experimental) or “light” (C12) (control) ICAT reagents prior to pooling, digestion, cation and then avidin chromatography. Quantitation is based on LC-MS peak areas of the stable isotope profiles of cysteine-containing peptides.

For locating cysteines involved in disulfide bonds, the heavy tag is used to label free cysteines prior to reduction/alkylation (iodoacetamide) of cysteines in disulfide bonds. The cysteine tag type indicates those involved in disulfide bonds.

only cysteine containing peptides are isolated.

The ICAT approach greatly simplifies a complex tryptic digest by looking at only the cysteine containing peptides.

Proteins with no cysteines will not be quantitated

since only a subset of the peptides are looked at, sites of post translational modification are lost in this technique.

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